The present invention relates generally to analytical test devices and, more particularly, to a method for increasing the dynamic measuring range of test elements based on specific binding reactions.
Immunological test strips are a widespread device for the rapid determination of drugs, pregnancy hormones, infectious diseases or so-called “cardiac markers” such as troponin T. In this connection qualitative tests that are read by purely visual means and often only yield a “yes-no” answer as well as quantitative tests which are evaluated by means of a reading instrument are widely used.
Rapid tests for immunologically detectable substances are known for numerous different parameters, for example from WO 97/06439, EP 0 291 194, U.S. Pat. Nos. 5,591,645, 4,861,711, 5,141,850, 6,506,612, 5,458,852, 5,073,484. In this case the immunological detection reagents (essentially labelled and unlabelled antibodies or antigens) are provided in a dry form on a support which allows the transport of a sample liquid (in particular body fluids such as blood, serum, plasma, urine, saliva, etc.) on or in the support. For this purpose the support is preferably capillary-active and is for example a membrane or a plastic support provided with capillary channels (such as, e.g., in U.S. Pat. No. 5,458,852). Among those skilled in the relevant art these are often referred to as immunological or immunochromatographic test strips or test devices. These terms as well as the term “carrier-bound immunological tests” or “carrier-bound immunological test elements” are often used synonymously and should also be interchangeable in the following.
In the case of simple systems and in particular in the case of purely qualitative analyses (where only the information “the analyte is present or not present” is of interest) such immunological test devices are often evaluated by purely visual means. This principle is now widely accepted in the market especially in the field of pregnancy tests.
(Semi)quantitative immunological rapid tests are usually evaluated with the aid of corresponding measuring instruments that are matched to the respective test strip. Different measuring principles are used depending on the type of labelling of the reagents of the test device used to detect the analyte. Optical detection methods and especially the measurement of reflectance and fluorescence are commonly used and simple to handle.
Many systems from the prior art ensure that the analyte detection zone (also abbreviated to “detection zone” in the following) and control zone are spatially narrowly delimited and arranged clearly separated from one another on the test device. For this purpose it has proven to be particularly advantageous to apply appropriate binding reagents in the form of lines or broken lines on the test device. Hence in order to evaluate the test device, spatially resolving optical systems such as for example camera chips or 2-dimensional or 3-dimensional photodiode arrays are often present in the measuring instrument for the purpose of evaluating the analyte detection zones and control zones. The signals of the optical systems are then converted by an appropriate evaluation software into concentration values and displayed.
With the immunological test devices of the prior art it is not possible to quantitatively detect any concentrations of the analyte in the sample. Towards the lower end, i.e., with regard to the lower detection limit, the measuring range is for example limited by the affinity and selectivity of the binding partners (usually antibodies) that are used and by the sensitivity of the detection optics which is limited with regard to the labels that are used. Saturation effects limit the measuring range towards the upper end, i.e., with regard to the dynamic measuring range. Thus in the case of analytes which can occur in very high concentrations in the sample, it is often not possible to provide an adequate amount of binding partner in the test device. In particular, in the analyte detection zones and control zones where the binding partners are arranged in a very restricted space on the test device, it is not possible to accommodate as much binding partner as would be desired. This can be particularly problematic in those cases in which a low detection limit for the analyte is required (and one therefore endeavors to concentrate the binding partners in the detection zone as strongly as possible, i.e., to accommodate them in a restricted space and thus due to the limited availability of binding sites on the test device only a relatively small amount of binding partner can be provided) but the analyte can be present in the sample in very variable amounts, i.e., very low as well as very high analyte concentrations can occur. At high analyte concentrations the detection zone is saturated with corresponding detection reagents resulting in a saturation behavior of the analyte concentration-detection signal relationship: The detection signal no longer increases above a certain analyte concentration, the evaluation curve levels off and can no longer be appropriately evaluated.
This is aggravated by the fact that especially with sandwich immunoassays not only is a levelling off of the curve to be observed at very high analyte concentrations which reflects the relationship between the analyte concentration and detection signal but also even a decrease of the signal with increasing analyte concentrations. This is referred to as the “high dose Hook effect”: At very high analyte concentrations it is observed that the signal intensity of sandwich immunoassays which initially increases as the analyte concentration increases, decreases again. This is explained by the fact that the amount of antibody offered in the test is no longer sufficient to form a sandwich complex (i.e., a complex comprising two antibodies per antigen) with the analyte molecules (antigens) in every case. There is an increasing formation of complexes consisting of analyte and in each case one antibody which, however, on their own are no longer detected. Thus measuring results that are false-negative or too low may occur which of course should be avoided.
Especially the quantitative immunological test strips in which a signal is determined by reflectance measurements, still have in some cases considerable weaknesses compared to conventional analytical systems that are usually used in large laboratories. In particular, the precision and the dynamic measuring range is usually worse in the case of test strips. This limits in particular the field of application of the highly sensitive sandwich assays for example for therapeutic monitoring where the largest possible measuring range is desired.
Moreover, for some parameters such as myoglobin or D-dimer a low detection limit is required, on the one hand, but on the other hand, very high concentrations of these analytes can occur in the sample material which are sometimes considerably above the decision limit “normal-pathological”. In these cases it would be desirable to have available test devices which have the largest possible measuring range in order to obtain reliable measured values without sample dilution. This would be of particular advantage for the use of such test devices to monitor the course of corresponding diseases.
In the prior art there has been no lack of concepts to solve the problems described above. However, up to now none of the proposals has been convincing in all points. In particular, the implementation of the concepts in the field of immuno-chromatographic test devices has not satisfactorily succeeded up to now.
U.S. Pat. No. 6,248,597 describes a heterogeneous agglutination immunoassay based on light scattering in which the dynamic measuring range is extended by mixing particles having different scattering properties. Binding partners having a high affinity for the analyte are immobilized on the particles which cause a large light scattering. In contrast, binding partners having a low affinity for the analyte are immobilized on the particles which cause a low light scattering.
A similar method is known from U.S. Pat. No. 5,585,241. In order to increase the dynamic measuring range, it proposes in connection with a flow cytometry immunoassay that two particles of different sizes are loaded with two antibodies having different affinities for the same antigen (small particles loaded with high-affinity antibody, large particles loaded with low-affinity antibody) and that an additional detectably-labelled antibody is used to detect the antigen by formation of a sandwich complex. The proposed system uses two different standard curves (one for each sort of particle) and allows a quantitative analyte determination by means of an ingenious software.
In order to avoid the Hook effect at high analyte concentrations (high dose Hook effect) U.S. Pat. No. 4,743,542 discloses a method in which, in addition to a detectably labelled antibody against the target antigen, a certain amount of the same but unlabelled antibody is simply added to the sample. As a result both antibodies compete for the analyte molecule and the oversaturation typical for the Hook effect only occurs, if at all, at higher antigen concentrations. As a result the dynamic measuring range is extended towards higher concentrations but at the expense of sensitivity. The use of low-affinity antibodies is proposed which have the same effect.
U.S. Pat. No. 4,595,661 describes heterogeneous sandwich immunoassays in which the Hook effect is avoided by using two soluble antibodies which have different affinities and specificities for the antigen, in addition to an immobilized capture antibody. The antibody having lower affinity only contributes significantly to the measuring signal at high antigen concentrations and hence prevents the Hook effect from becoming noticeable.
It is known from U.S. Pat. No. 5,073,484 that an immunologically detectable analyte can be quantitatively detected using several discrete, successive binding zones in a flow-through support. The number of zones in which the specific binding and detection reactions take place increases with an increasing amount of analyte in the sample. The number of zones which are colored after sample contact correlates with the amount of analyte in the sample. U.S. Pat. No. 5,073,484 proposes that the number of binding zones be increased in order to increase the accuracy and to extend the measuring range. A disadvantage of this is that an automatic evaluation of the binding zones requires a relatively complicated optical system which is able under certain circumstances to simultaneously detect and evaluate a large number of zones in order to thus allow a quantitative analyte determination. Moreover, the test devices have to be relatively long due to the relatively large number of discrete binding zones that are spatially separated from one another. Thus in order to ensure that the sample reliably migrates through the test device, it is necessary to use relatively large sample volumes which especially if it is intended to use whole blood samples, is also disadvantageous especially for reasons of sample collection.
WO 00/31538 describes immunochromatographic test strips in which one or more control zones are accommodated on an absorbent matrix in addition to an analyte detection zone. Binding partners for the analyte provided with a detectable label are bound to the matrix in the analyte detection zone as well as in the control zones. In this process exactly defined amounts of labelled binding partner are bound in the control zones where these amounts are independent of the amount of analyte in the sample. Different amounts of labelled binding partner are preferably bound in the control zones such that quasi internal comparative scales are present on the test strip. The control zones are used for calibration when evaluating the analyte detection zone. In order to increase the dynamic measuring range especially for non-linear concentration-measuring signal relationships, WO 00/31538 proposes that additional control zones are provided on the test strip.
In the case of immunochromatographic test strips which use fluorescent labels for analyte detection, it is known from J. Hampl et al., “Upconverting Phosphor Reporters in Immunochromatographic Assays”, Analytical Biochemistry 288, 176-187 (2001) that the control zone (control line) which contains a species-specific antibody in an immobilized form can also be used to evaluate the measured signal in addition to the actual detection zone (target line) which contains an analyte-specific antibody in an immobilized form. A similar use is described by OraSure Technologies Inc., Bethlehem, Pa., USA, on www.orasure.com. The evaluation of the target line as well as control line is primarily used to eliminate variations in the measured signal that are due to the actual amount of liquid in the optically measured region of the test strip. As a result the sensitivity of the detection method (assay) is also indirectly increased (the dynamic measuring range is thus extended towards lower concentrations). In contrast an extension of the dynamic measuring range towards higher concentrations is not reported.
The dynamic measuring range of immunochromatographic test devices can also be de facto extended by diluting the sample material accordingly before analysis. An extension of the measuring range achieved in this manner is unsatisfactory since it requires additional handling steps that could potentially lead to errors in the analysis. Furthermore, especially in cases in which an analyte could occur in very high as well as in very low concentrations in similar samples, a controlled sample dilution is only advisable when the analyte is present in the sample at high concentrations but not in the reverse case since otherwise the concentration may fall below the lower detection limit as a result of the dilution and the analyte may be falsely not detected in the sample.
There has been previously a lack of simple and reliable methods for extending the dynamic measuring range of immunochromatographic test devices towards higher analyte concentrations without adversely affecting the lower detection limit for the analyte detection.